Current Proteomics - Volume 15, Issue 1, 2018

Therapeutic proteins can be pharmacologically improved by conjugative post-translational modifications through the rational design of their structure and production process. N- and O- linked glycosylation can confer advantages to proteins and can be controlled by the process conditions, producing cell lines and enzymatic expression or activity. The resulting glycan profile influences their pharmacological features such as in the biopharmaceutical velaglucerase alfa. Conjugation of polyethylene glycol onto therapeutic proteins has been used to overcome pharmacological limitations. They can be site-specifically or randomly linked through the reaction between a terminal group of the polyethylene glycol molecule and an amino acid residue group of the protein. Both components (protein and polyethylene glycol) participate in pharmacological mechanisms, enabling a longer half-life for peginterferon beta-1a, for example. Fatty acylation of proteins occurs in cells via some known reactions that may involve different linkages, fatty acids, modified residues, and enzymes. Insulin degludec is a fatty acylated biopharmaceutical formulated to solve the problem of variability in insulin exposure associated with other products. Conjugative post-translational modifications can be used to pharmacologically improve even therapeutic proteins produced by prokaryotic cell lines while exploring them may result in the development of other potential uses as well as new generations of biopharmaceuticals.

Background: One of the most fundamental problems, which remain unsolved, is how entirely new gene (EntNew gene) was produced, which is totally different gene from any previously existing gene. On the other hand, we have proposed GC-NSF(a) hypothesis on formation of EntNew gene/protein, assuming that EntNew gene is generated from non-stop frame on antisense strand of GCrich gene (GC-NSF(a)). Objective: The objective of this study is to obtain direct evidence clearly supporting the GC-NSF(a) hypothesis through computer analysis of gene/protein databases of GC-rich microbial genomes. Method: In order to get direct evidence for the hypothesis, every base sequence encoded by GC-rich P. aeruginosa PAO1 genome (GC content=66.6%) was transformed into antisense sequence (GCNSF( a)). NSF(a) is a non-stop frame codon sequence on antisense strand in the reading frame corresponding with gene on sense strand. AAS of every imaginary protein encoded by the GC-NSF(a) was homology-searched against all extant proteins encoded by the same genome. Program version 2.2.30+ of BLASTP in NCBI for computational investigation was used in the homology-search. Results: From the results, it was found that AAS encoded by GC-NSF(a) of tal gene has sufficient homology with AAS encoded by ftsZ gene. In addition to another result from the P. aeruginosa genome, three cases showing sufficient homology between AAS encoded by GC-NSF(a) and AAS of extant protein were also obtained from the results similarly analyzed with 57 GC-rich microbial genomes. Conclusion: Thus, we conclude that EntNew gene encoding EntNew protein has been generated from GC-NSF(a), according to the GC-NSF(a) hypothesis.

Background: Prokaryotic systems such as E. coli are among the most affordable and simplest hosts which are being employed to express recombinant proteins, nevertheless without appropriate signal peptide these systems cannot be used for secretory proteins. Bovine pancreatic ribonuclease A is a protein with four disulfide bonds which might be used as an immunoenzyme for immunotherapy. Consequently, the production of this recombinant protein, using prokaryotic system, requires a suitable signal peptide to protect disulfide bonds and to prevent misfolding. Objective: This study was designed to predict the best signal peptides to express bovine pancreatic ribonuclease A protein in E. coli. Method: In this study, 42 signal sequences were selected from data bases and the most important features of them were evaluated. First, n, h and c regions of signal peptides and their probability were investigated by signalP software’s version 4.1. Then, physico-chemical features of them were evaluated by Portparam and SOLpro. Also, secretion sorting and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB software programs. Results: The results showed that among all studied signal peptides only 28 out of 41 remained signal peptides could be considered as appropriate secretory signal peptides. Conclusion: Finally, Phage shock protein E, ranked as the best signal peptide and after that, pectate lyase B, F41 fimbrial protein and Lipopolysaccharide export system protein lptA considered as the next best signal peptides which were approved by in silico tools as the most appropriate secretory signal peptides in E. coli. However, further experiments are required to validate these in silico results.

Background: Pulmonary embolism (PE) is a cardiovascular emergency that affects many patients. Acute PE-induced oxidative stress can lead to the accumulation of nitroproteins that may play a role in disease progression. The impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, related to pathological conditions. Methods: We used a proteomic approach to analyze nitrated plasma proteins in patients diagnosed with acute PE. Nitrotyrosine (NO2Tyr)-containing proteins were immunoprecipitated with a NO2Tyr affinity sorbent. Precipitated proteins were separated by SDS-PAGE and visualized by either Coomassie Blue staining or western blotting with mouse monoclonal anti-NO2Tyr antibody. Immunoreactive bands observed in disease patients were in-gel digested and analyzed by MALDI-TOF mass spectrometry (MS). Results: Mass fingerprint data sets obtained from the 138 kDa peptide fragment ions matched human collagen alpha-1 (III) chain (CO3A1) with Mascot algorithm analysis giving a score of 65 (p< 0.05). Mass fingerprint data sets obtained from the 250 kDa peptide fragment ions matched human chondroitin sulfate proteoglycan 4 (CSPG4) with Mascot algorithm analysis giving a score of 57 (p< 0.05). Nitration-induced alterations of CSPG4 activity can thus possibly lead to decreased fibrin degradation and enhanced complement system activity. Conclusion: In vivo characteristics of these nitroproteins could be significant with regards to biomarker studies and understanding of disease mechanism in patients with PE. Future studies are aimed to understand the relevance of NO2Tyr modifications in CO3A1 and CSPG4 relating to changes in protein structure and function.

Background: Several bacteria tolerate chromate with this attribute important in bacteria intended for biotechnological applications on chromate clean-up. Marine bacteria are beginning to show promise in this field due to their range and novelty. However, the general mechanisms of chromate tolerance employed by marine bacteria remain unknown. Objective: To understand differential protein expression of a marine Staphylcoccus sp. in terms of response to hexavalent chromium stress. Method: In this study, protein extracted from Staphylococcus xylosus grown in varying chromate (Cr (VI)) conditions (50, 100, 150 and 200 μg mL-1 Cr (VI)) was subject to spectral counting strategy where unlabelled protein samples were analyzed separately using the same protocol as each other, and the relative protein quantification was established by comparing the number of MS/MS spectra identified for each protein. Results: A total of 613 proteins detected in S. xylosus were differentially expressed when grown in varying Cr (VI) conditions and were grouped functionally. Response in medium with 50 μg mL-1 Cr (VI) was not very different from that of the control (no Cr). Whereas, in medium with 200 μg mL-1 Cr (VI), the ability of S. xylosus to cope with Cr stress seemed to be reduced as the number of upregulated stress tolerant proteins (4) was less than those observed in medium with 150 μg mL-1 (15) or 100 μg mL-1 Cr (VI) (16). Up-regulation patterns showed that superoxide removal, directed DNA repair and proteins involved in peptide mis-folding were mechanisms employed by S. xylosus for surviving Cr induced stress in medium with 100 and 150 μg mL-1 Cr (VI). Conclusion: These findings have important implications for understanding mechanisms of chromate tolerance, which provides new information of how a marine Staphylococcus deals with chromate toxicity.

Background: EGCG, (-)-epigallocatechin-3-gallate, a constituent of catechin with the highest content in green tea, is currently show anti-atherogenic effects ex vivo in humans. Objective: To study the effect of EGCG on proteome of Raw264.7 macrophage-derived foam cell by using two-dimensional electrophoresis and mass spectrum. Method: The proteins from macrophage cells, foam cells, and EGCG-treated cells with 30μg/mL EGCG were separated with two-dimensional electrophoresis and the gels were scanned after silver staining. All images were analyzed by Image Master 2D 6.0 software, and the different proteins were identified by mass spectrum. Results: M2-type pyruvate kinase, glucose-6-phosphatase, triose phosphate isomerase, ATP synthase, stathmin 1, proteasome 26S, ubiquitin-conjugating enzyme, ring finger protein 130, glyoxalase domain containing 4, beta-actin, Tropomyosin, A-X actin, heat shock protein 70, Adenine phosphoribosyltransferase, macrophage migration inhibitory factor 13kda protein, and anti-sense basic fibroblast growth factor, were identified to be differentially expressed among the macrophage cells, foam cells, and EGCG-treated foam cells. Conclusion: EGCG prevents the development of atherosclerosis probably through changing the expression of cytoskeletal proteins and antioxidant enzymes to regulate the energy metabolism in foam cells.

Background: In many human diseases protein kinase are known to play a central role. Protein kinases phosphorylate its substrates and they themselves regulated through phosphorylation of their activation loop and become catalytically active. LMTK3 is an oncogenic protein kinase, implicated in breast cancer progression and endocrine resistance. Recent report says phosphorylation of LMTK3 by CDK5 results in breast cancer tumor progression. Thereby information about interface residues and probable phosphorylation site on LMTK3 is critical. Objective: To understand the transient protein – protein interactions between CDK5 and LMTK3 using computational techniques. Methods: LMTK3 structure was superimposed with known kinases to determine the probable activation segment and phosphorylation sites in LMTK3. PatchDock was used to obtain CDK5-LMTK3 complex structure. PDBsum server was used to identify the interface residues between CDK5 and LMTK3. The stability of CDK5-LMTK3 complex was studied using Molecular dynamics (MD) simulation. Results: From PatchDock, interface area between CDK5-LMTK3 complex was found to be 2081 Ų with atomic contact energy of -228.80 kcal/mol. PDBsum result reveal that, CDK5 interact and displayed non-bonding interactions with the probable phosphorylation sites of LMTK3. Total number of interface residues across CDK5-LMTK3 was found to be around 50 and the interface area found to be 1274 Ų (in CDK5) and 1224 Ų (in LMTK3). From MD simulation, CDK5-LMTK3 complex was found to be stable. Conclusion: This study enhances the understanding of interactions between CDK5 and LMTK3 that may be helpful in understanding the LMTK3 phosphorylation by CDK5 which is considered to be a new cellular pathway in breast cancer tumor progression.

Background: Human IFN-α is a family of structurally related proteins that exhibit a wide range of antiproliferative and antiviral activities. IFNs bind to a specific cell receptor as the IFN-α/β receptor (IFNAR) that compose of IFNAR1 and IFNAR2 chains. Interferon’s affinity for IFNAR1 and IFNAR2 chains changes the type of its activity. Objective: To understand the structural basis for these different activities, some important amino acid residues in this study binding region of interferon to IFNAR1 were replaced to more effective ones. Method: In order to suggestion of the important amino acids for mutation, the imperative area IFN- α were distinguished based on previous reports and structural properties including Vander Waals energy, electrostatic energy, length of the hydrogen bands and other biological property of selected and substituted amino acids. Both the IFN-α2b and its receptor IFNAR1 have been modeled followed by Easy Modeler2 software and all of the structures were confirmed by server MDWEB and VMD software. Then, the interaction studies between two protein have been done using protein-protein docking (HADDOCK 2.2) for both native and mutant forms. Results: HADDOCK results for investigation of IFN-α2b and IFNAR1 interactions in both native and mutant forms shown that binding of mutant forms to IFNAR1 have been enhanced. Conclusion: In silico study of IFN mutants have provided a powerful tool for studying the structure and function of these molecules. Moreover, these engineered IFNs may have important new therapeutic applications and may provide more sights into understanding of the clinical activities of these molecules.

Objective: Secreted proteins from fibroblasts critically contribute to tumor microenvironment. Human embryonic lung fibroblast MRC-5cell line is a widely used model in cancer formation and progression studies. However, data regarding its secretome are poor. Method: In the present study, we used immobilized pH gradient-based two dimensional gel electrophoresis (2DE) and mass spectrometry to characterize the secretome of MRC-5 cell line. Results: In total, 21 protein spots related to 17 proteins were identified. Some of them were insulin-like growth factor-binding protein 5, epididymal secretory protein E1 (Niemann-Pick disease type C2 protein), cathepsin L, glutathione S-transferase P, ubiquitin carboxyl-terminal hydrolase isozyme L1 (PGP9.5), UPF0587 protein C1orf123, transgelin, cofilin-1, cofilin-2, and guanine nucleotide-binding protein subunit beta-2-like 1. Conclusion: Using 2DE and mass spectrometry, we identified several proteins in the conditioned media of MRC-5 cell line. The identified proteins were known to be involved in the key cellular processes. Our findings shed more light on the proteome mapping of secreted proteins from a fibroblast cell line.